The goal of this Phase II proposal is to evaluate Scarab Genomics'improved E. coli production strains in "real world" conditions under which pharmacological grade therapeutic proteins and plasmid DNA are produced. In some cases this will be done in collaboration with contract manufacturers using GMP facilities. Candidate proteins and plasmids will be obtained through collaborations with biotech companies which are developing pharmaceuticals that could be manufactured with Scarab's Clean Genome(r) E. coli strains. Since 1982, when the FDA approved human insulin produced by E. coli for marketing, E. coli fermentation has been a preferred and cost-effective method for commercial production of therapeutic DNA and proteins. Many innovations have been made to the expression systems used with E. coli, but the actual bacteria used commercially had not been basically improved since the 1970's until Scarab's scarless genomic deletion technology made it possible to systematically revise the genome of E. coli. This technology has enabled Scarab to create "reduced genome" strains of E. coli that lack many genes (up to 20% of the chromosome) that are not beneficial for commercial applications. The "clean genome" strains have improved metabolic efficiency and product yield, increased clone stability and have eliminated many undesired fermentation side products including phage, flagellae, fimbrae, transposases and in some cases, toxins. Phase I of this project dealt with the problem of limiting the most serious E.coli toxin, endotoxin (also called lipopolysacharide or LPS), which is present at full strength in all currently used E. coli production strains. The FDA places strict limits on endotoxin levels in pharmaceuticals. Endotoxin cannot be completely eliminated because the genes coding for the core LPS structure are essential for growth, but discoveries made in Phase I led to genetic mutations reducing the level of endotoxin substantially. The best of our low endotoxin E. coli, when injected into mice, decreases mortality by more than 10-fold over the ordinary E. coli currently used in production. It is hoped that this Phase II project will lead to an FDA application for clinical trial of at least one pharmaceutical product produced by a Scarab strain from one of our collaborators. A demonstration that Scarab's strain has been FDA approved will remove a significant barrier to our marketing, namely the conservative perception that what has been approved before is good enough. PUBLIC HEALTH RELEVANCE: Plasmid DNA and protein generated from bacterial fermentation is subject to stringent purity constraints because of the potential for contamination of samples with impurities that include endotoxin. Because of the health risk of therapeutics that contain impurities like endotoxin and due to the high cost of purification, generating bacterial strains that reduce impurity carryover and toxicity are of tremendous value to the biologic and pharmaceutical industry.